Despite their clonal origin cells from advanced carcinomas are often genetically heterogeneous Tumor cell variability is thought to result from genomic instability Clonal heterogeneity in turn predicts a poor outcome, and resistance to therapy. We propose that in thyroid tumors, the nature of the oncogenic events involved in the initiation of the neoplastic clone may determine the likelihood of genomic instability occurring at a later stage. Among the various forms of thyroid neoplasia follicular carcinomas are commonly aneaploid, whereas abnormalities in chromosome number are comparatively less frequent in papillary thyroid carcinomas Mutations of Ras are most prevalent in benign and malignant follicular neoplasms, and believed to be one of the early steps in thyroid tumor formation. In this proposal we will test the hypothesis that tumor initiation by constitutively activated Ras predisposes thyroid cells to chromosomal instability, and will explore the effector systanu involved Acute expression of oncogenic Ras in thyroid cells pre-synchronized at the G1/S boundary accelerates passage through G2/M, and causes defective DNA damage and mitotic spindle assembly checkpoints. This is followed by chromosome misalignment, multiple spindle formation, centrosome amplification and generation of micronuclei. Although constitutive activation of MAP kinase is a mediator of chromosomal instability in this model, the possible role of other effector pathways of Ras is unknown The following aims are proposed: 1) The relative contribution of the Raf-MEK-ERK compared to alternative effectors on cell cycle progression through G2/M, checkpoint function and chromosomal instability will be determined by doxycycline-induced expression of H-Ras effector domain mutants that either signal selectively through Raf (V12S35), or are unable to do so (V12C40; V12S37). The Raf dependency of the effects will be further tested after acute expression of a constitutively activated Raf1 (Raf-BXB), or of a mutant of Raf-BXB that does not activate MEKs 2). We will determine whether inappropriate MEK activation mediates Ras disruption of the DNA damage response, and define whether MEK2 is preferentially involved. We will also characterize MEK2 interacting proteins with which it associates during (32 arrest 3) The biological significance of oncogenic H-ras effects on genomic stability will be determined in vivo in mice with a thyroid-specific knock-in of H-ras V12 4) We will establish whether anenploidy in thyroid neoplasms is associated with a high prevalence of Ras mutations Although Ras mutations and aneuploidy are more prevalent in follicular neoplasms than in papillary carcinomas, this association has not been rigorously established. This would be consistent with the notion that Ras mutation, in thyroid cells induce a "mutator" phenotype, and are thus more likely to undergo phenotypic progression.